Program Director/Principal Investigator (Last, First, Middle): Geballe, Adam P. Project Summary/Abstract Human cytomegalovirus (HCMV) is a widespread pathogen that causes life-threatening diseases in immunocompromised patients and long-term neurological disability in congenitally infected newborns. In addition to its medical importance, HCMV and related mammalian CMVs provide a powerful system for uncovering insights into the mechanisms and evolution of host-viral interactions the enable or thwart the ability of viruses to control the host cell protein synthetic machinery. These studies focus the broadly acting antiviral pathway mediated by the host restriction factor protein kinase R (PKR). During the next grant period, efforts will be directed to elucidating how the HCMV protein TRS1 associates with PKR to block its function. Aim 1 will investigate the potential role of a dsRNA bridge linking the amino-termini of the proteins using pull down and immunoblot assays of proteins expressed in cell free systems and in cells infected with wild type and mutant viruses. The role of carboxy-terminal species-specific hydrophobic interactions of TRS1 and PKR will be revealed by studies of chimeric and mutant proteins using functional and binding assays in human cells and in yeast. Aim 2 will employ a powerful newly emerging technique, deep mutational scanning, to uncover the function of an intriguing rapidly evolving region of PKR that interacts with its primary substrate, eukaryotic initiation factor 2? (eIF2?) and is targeted by diverse viral antagonists. The role of this same PKR domain in nonhuman primate PKR alleles as a target of TRS1 gene homologs in Old World and New World monkey CMV will be determined. Aim 3 will test whether the TRS1 is essential in order to block the eIF2? kinase or another function of PKR using pharmacologic and genetic tools. Finally, studies of the impact of activating the integrated stress responses on replication of wild type and mutant viruses in wild type and knock-out cells will reveal whether TRS1 influences other cellular eIF2? kinases. Together these studies will clarify critical structural and functional relationships in the TRS1-PKR-eIF2? pathway that regulate CMV replication.